Regulatory T (Treg) cells play a central role in maintaining immune homeostasis. However, little is known about the stability of Treg cells in vivo. In this study, we demonstrate that a significant percentage of cells exhibited transient or unstable Foxp3 expression. These exFoxp3+ T cells express an activated-memory T cell phenotype, and produced inflammatory cytokines. Moreover, exFoxp3 cell numbers increased in inflamed tissues under autoimmune conditions. Adoptive transfer of autoreactive exFoxp3 cells led to the rapid-onset of diabetes. Finally, T cell receptor repertoire analyses suggested that exFoxp3 cells develop from both natural and adaptive Treg cells. Thus, the generation of potentially autoreactive effector T cells as a consequence of Foxp3 instability has important implications for understanding autoimmune disease pathogenesis.

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The prevention of autoimmunity requires elimination of self-reactive T cells during their development and maturation. Expression of diverse self-antigens by stromal cells in the thymus is essential to this process, and depends, in part, on the activity of the Autoimmune Regulator (Aire) gene. Here we report the identification of extrathymic Aire-expressing cells (eTACs) resident within the secondary lymphoid organs. These stromally-derived eTACs express a diverse array of unique self-antigens and are capable of interacting with and deleting naïve autoreactive T cells. Using twophoton microscopy we observe stable, antigen-specific interactions between eTACs and autoreactive T cells. We propose that such a secondary network of self-antigen-expressing stromal cells may help reinforce immune tolerance by preventing the maturation of autoreactive T cells that escape thymic negative selection.

A new regulatory T (T reg) cell–specific, FoxP3-GFP-hCre bacterial artificial chromosome transgenic mouse was crossed to a conditional Dicer knockout (KO) mouse strain to analyze the role of microRNAs (miRNAs) in the development and function of T reg cells. Although thymic T reg cells developed normally in this setting, the cells showed evidence of altered differentiation and dysfunction in the periphery. Dicer-deficient T reg lineage cells failed to remain stable, as a subset of cells down-regulated the T reg cell–specific transcription factor FoxP3, whereas the majority expressed altered levels of multiple genes and proteins (including Neuropilin 1, glucocorticoid-induced tumor necrosis factor receptor, and cytotoxic T lymphocyte antigen 4) associated with the T reg cell fingerprint. In fact, a significant percentage of the T reg lineage cells took on a T helper cell memory phenotype including increased levels of CD127, interleukin 4, and interferon γ. Importantly, Dicer-deficient T reg cells lost suppression activity in vivo; the mice rapidly developed fatal systemic autoimmune disease resembling the FoxP3 KO phenotype. These results support a central role for miRNAs in maintaining the stability of differentiated T reg cell function in vivo and homeostasis of the adaptive immune system.

Stable Foxp3 expression is crucial for regulatory T (Treg) cell function. We observed that antigen-driven activation and inflammation in the central nervous system (CNS) promoted Foxp3 instability selectively in the autoreactive Treg cells that expressed high Foxp3 levels before experimental autoimmune encephalitis induction. Treg cells with a demethylated Treg cell-specific demethylated region in the Foxp3 locus down-regulated Foxp3 transcription in the inflamed CNS during the induction phase of the response. Stable Foxp3 expression returned at the population level with the resolution of inflammation or was rescued by IL-2:anti-IL-2 complex treatment during the antigen priming phase. Thus, a subset of fully committed self-antigen-specific Treg cells lost Foxp3 expression during an inflammatory autoimmune response and may be involved in inadequate control of autoimmunity. These results have important implications for Treg cell therapies, and give insights into the dynamics of the Treg cell network during auto-reactive CD4+ T cell effector responses in vivo.

SummaryRegulatory T (Treg) cells play an essential role in maintaining immunological tolerance. The discovery of FoxP3 as a key Treg transcription factor combined with recent advances in the development of functional reporter mice have enabled new insights into Treg biology and revealed unexpected features of this lineage. In this review, we address the stability of this population, focusing on studies that suggest that Tregs can down-regulate FoxP3, lose regulatory activity and, under some conditions, become memory T cells capable of recognizing self-antigens and expressing effector cell activities including the production of IL-17 and IFNγ. The presence of these "exTregs" in multiple inflammatory settings suggests a potential role for these cells in a variety of disease settings ranging from autoimmunity to cancer and infectious disease.

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Regulatory T (Treg) cells are essential for self-tolerance and immune homeostasis. Lack of effector T cell (Teff) function and gain of suppressive activity by Treg are dependent on the transcriptional program induced by Foxp3. Here we report repression of SATB1, a genome organizer regulating chromatin structure and gene expression, as crucial for Treg phenotype and function. Foxp3, acting as a transcriptional repressor, directly suppressed the SATB1 locus and indirectly through induction of microRNAs that bound the SATB1 3′UTR. Release of SATB1 from Foxp3 control in Treg caused loss of suppressive function, establishment of transcriptional Teff programs and induction of Teff cytokines. These data support that inhibition of SATB1-mediated modulation of global chromatin remodelling is pivotal for maintaining Treg functionality.